Microfluidic device

ABSTRACT

Described herein are particular embodiments relating to a microfluidic device that may be utilized for cell sensing, counting, and/or sorting. Particular aspects relate to a microfabricated device that is capable of differentiating single cell types from dense cell populations. One particular embodiment relates a device and methods of using the same for sensing, counting, and/or sorting leukocytes from whole, undiluted blood samples.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §120 asa continuation of U.S. patent application Ser. No. 14/685,480, filedApr. 13, 2015, and now U.S. Pat. No. 9,234,884, which is a continuationof U.S. patent application Ser. No. 14/296,199, filed Jun. 4, 2014, andissued as U.S. Pat. No. 9,029,158 on May 12, 2015, which is a divisionalof U.S. patent application Ser. No. 12/062,808, filed Apr. 4, 2008, nowabandoned, which claims priority under 35 U.S.C. §119(e) to U.S.provisional patent application No. 60/922,296, filed Apr. 6, 2007, nowexpired, the contents of each of which are herein incorporated byreference in their entirety.

GOVERNMENT RIGHTS

This invention was made with government support under grant number NCC9-58-317 awarded by National Space Biomedical Research Institute throughNASA. The government has certain rights in this invention.

FIELD OF THE INVENTION

The present disclosure relates to fabricated microfluidic devices thatcan be utilized as cell sensors and/or actuators. In certainembodiments, the microfluidic device may be used for labeling, sensing,differentiating, and/or sorting targets, particularly cell populations.

BACKGROUND OF THE INVENTION

Standard cell sensors or actuators are generally based on flow cytometryand employ one or a combination of electrical impedance sensing, lightscattering measurement, and chemical or immunostaining followed byoptical sensing.

For differentiation of blood cells by electrical impedance sensing, redblood cells are removed by lysing in order to reduce the blood volume.Lysing is generally done through the use of saponin or surfactants.During the lysing process, the leukocyte cell volume changes dependingon cell type, due to the leakage of cytoplasm contents and cell nucleusshrinkage in varying amounts. Fujimoto, Sysmex J. Int. 9 (1990). Thus,normally 2-part (lymphocytes versus granulocytes) or even 3-part(lymphocytes, neutrophils, and other leukocytes) differential can beachieved by simple electrical impedance measurement of particle volume.Hughes-Jones, et al., J. Clin. Pathol. 27; 623-625 (1974); Oberjat, etal., J. Lab. Clin. Med. 76; 518 (1970); Vandilla, et al., Proc. Soc.Exp. Biol. Med. 125; 367 (1967); Maeda, et al., Clin. Pathol. 27;1117-1200 (1979); Maeda, et al., Clin. Pathol. 9; 555-558 (1982).Combining direct current and alternating current impedance, specialacidic hemolysis in basophile channel and alkali hemolysis in eosinophilchannel, a 5-part leukocyte differential can be achieved. Tatsumi, etal., Sysmex J. Int. 9; 9-20 (1999).

Alternative optical methods are based on light scattering andfluorescence staining of organelles, granules, and nuclei. Generally,low-angle scattered light contains information on cell size andhigh-angle scattered light can be used to probe internal composition ofthe cell. To achieve 5-part differential, certain leukocyte populations,such as eosinophils, require special stain to change its scatteringcharacteristics from other granulocytes, and basophils typically need tobe counted separately following the differential lysis of otherleukocytes. McKenzie, Clinical Laboratory Hematology, Prentice Hall,2004; Fujimoto, Sysmex J. Int. 9 (1999).

In general, conventional automated cell analyzers are bulky, expensive,and mechanically complex, which restricts their locations to hospitalsor central laboratories. Conventional cell analyzers require largersample volumes and generate more waste than the systems developed usingmicrodevices. Furthermore, for analysis of certain cell types, such asleukocytes, accuracy and speed of counting, differentiation, and/orsorting is important for determining disease state and treatment.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS(S)

FIG. 1A shows the molecular structure of acridine orange.

FIG. 1B shows leukocyte staining results with acridine orange.

FIG. 2 shows the top view of one embodiment of a novel fabricatedmicrofluidic apparatus.

FIG. 3 shows one embodiment of the optical system setup.

FIG. 4A shows erythrocyte concentration in whole blood.

FIG. 4B shows leukocyte staining in whole blood with acridine orange ata concentration of 100 ng/mL.

FIG. 4C shows leukocyte staining In whole blood with acridine orange ata concentration of 1 μg/mL.

FIG. 4D shows leukocyte staining in whole blood with acridine orange ata concentration of 10 μg/mL.

FIG. 4E shows leukocyte staining In whole blood with acridine orange ata concentration of 100 μg/mL.

FIG. 4F shows leukocyte staining in whole blood with acridine orange ata concentration of 1 mg/mL.

FIG. 5 shows fluorescent signal bleaching from a single leukocyte in oneembodiment.

FIG. 6A shows an image of background and a 5 μm bead with focused laserillumination flow taken by CCD camera with long pass emission filter,according to one embodiment.

FIG. 6B shows an image of background and a 5 μm bead with laserillumination flow taken by CCD camera with long pass emission filter,according to one embodiment.

FIG. 6C shows an image of background and a 5 μm bead with diffused laserillumination flow taken by CCD camera with long pass emission filter,according to one embodiment.

FIG. 6D shows an image of background and the trace of a 5 μm bead withdiffused laser illumination flow taken by CCD camera with long passemission filter, according to one embodiment.

FIG. 7 shows a graph of detection of 5 μm fluorescent beads detectionwith photo-diode detector with long pass emission filter.

FIG. 8A shows background image of focused laser beam from video taken byCCD camera with long pass emission filter, according to one embodiment.

FIG. 8B shows a signal from a leukocyte from diluted whole blood testingfrom video taken by CCD camera with long pass emission filter, accordingto one embodiment.

FIG. 9 shows the lime trace of amplified photodiode signal of acridineorange stained undiluted whole blood with green emission filter centeredat 525 nm, and peaks labeled, according to one embodiment.

FIG. 10 shows a histogram of signal intensity from photodiode detectorwith green emission filter centered at 525 nm.

FIG. 11 shows a histogram of signal intensity from photodiode detectorwith red emission filter centered at 650 nm.

FIG. 12A shows an illustration of a handheld defection box instrumentaccording to one embodiment.

FIG. 12B shows an assembled detection box instrument according to oneembodiment.

FIG. 13A shows the top view of an apparatus according to one particularembodiment.

FIG. 13B shows the top view of an apparatus according to one particularembodiment.

SUMMARY OF THE INVENTION

Certain embodiments disclosed herein include a microfluidic apparatuscomprising a substrate having a first channel having a defined physicalfeature, wherein said first channel is in fluid communication with atleast one inlet for receiving a fluid, wherein said first channel leadsto a restrictive access, and wherein said first channel is in fluidcommunication with a second channel having a defined physical feature,wherein said second channel is in fluid communication with at least onefluid flow outlet; and a fluid biological sample. In certainembodiments, said defined physical feature is a depression orprotrusion. In particular embodiments, said fluid biological samplecomprises blood. In certain embodiments, the microfluidic apparatusfurther comprises a detection zone, and/or a filter array, each in fluidcommunication with said channel and said fluid flow outlet.

A microfluidic apparatus comprising a substrate having at least onefirst channel having a defined physical feature; at least one firstinlet formed in said first channel for receiving a first fluid; whereinsaid first channel is in fluid communication with a bifurcated channel,wherein said bifurcated channel is in fluid communication with a thirdchannel detection zone; at least one second inlet for receiving a secondfluid, wherein said second inlet is in fluid communication with abranched channel; a filter structure in fluid communication with areservoir, wherein said reservoir is in fluid communication with saidthird channel detection zone; at least one fluid flow outlet formed Insaid third channel; and a fluid sample; wherein the ratio of thecross-sectional area of said second channel compared to thecross-sectional area of said first channel is 1:10. In certainembodiments, the defined physical feature is a depression or aprotrusion.

In certain embodiments, said filter structure comprises a filter array,said first fluid comprises sheath fluid and said second fluid comprisesblood.

Certain embodiments disclosed herein relate to a detection systemcomprising a microfluidic apparatus and further comprising a lightsource; a lens assembly; a filter assembly; and an image capture device.In some embodiments, the detection system further comprises at least onedisplay unit or at least one recording unit. In certain particularembodiments, said excitation source comprises a laser, particularly anargon laser. In particular embodiments, said filter assembly comprisesan excitation filter, and at least one emission filter. In certainembodiments, said filter assembly further comprises at least oneaperture and at least one neutral density filter. In particularembodiments, said filter assembly further comprises at least one glasspolarizer.

In certain embodiments, the lens assembly of the detection systemcomprises at least one condenser lens, at least one objective lens, andat least one beamsplitter. In particular embodiments, said image capturedevice comprises at least one CCD camera, CMOS device, photodiode, orphotomultiplier tube. In certain embodiments, said filter assemblycomprises at least two emission filters and said image capture devicecomprises at least one photomultiplier tube. In certain embodiments,said display unit comprises a computer and said recording unit comprisesan oscilloscope. In particular embodiments, said excitation sourcecomprises an argon laser; said lens assembly comprises a condenser lens,an objective lens, and a beamsplitter; said filter assembly comprises anexcitation filter, a pinhole aperture and a neutral density filter, andat least one emission filter; said image capture device comprises a CCDcamera and a photodiode, and said display unit comprises a personalcomputer, and further comprising an amplifier.

In particular embodiments, said excitation source of the detectionsystem comprises an argon laser; said lens assembly comprises acondenser lens, an objective lens, and a beamsplitter, said filterassembly comprises an excitation filter, at least one emission filter;said image capture device comprises a photomultiplier tube, and saiddisplay unit comprises a personal computer.

Other embodiments disclosed herein relate to a method for identifying atarget comprising providing a fluid sample to at least one microfluidicapparatus, wherein said fluid sample contains at least one dye;providing an excitation source to induce at least one fluorescent signalin a target; defecting the fluorescent signal using a sensor in theapparatus; and identifying the target based in part on the analysis ofthe fluorescent signal. In certain embodiments, said target is selectedfrom the group consisting of: cells, organelles, nuclei, granules, DNA,and RNA. In other embodiments, said target comprises a cell selectedfrom the group consisting of a monocyte, a granulocyte, a macrophage, aneutrophil, an eosinophil, a basophil, or other leukocyte. In specificembodiments, said target comprises a leukocyte and said dye comprisesacridine orange. Particular embodiments of the method further comprisingcounting or sorting the target in the sample by analysis of thefluorescent signal. In certain embodiments, said fluid sample comprisesblood.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to fabricated microfluidic devices thatcan be utilized as cell sensors and/or actuators. In certainembodiments, the microfluidic device may be used for labeling, sensing,differentiating, and/or sorting cell populations.

Microfluidic cell sensors and actuators can provide cell sensing andcounting for a more accurate outcome and a lower cost. Particle counting(including bead, erythrocyte, and cultured cell) has been demonstrated,for example, by electrical impedance sensing, light scatteringdetection, and fluorescent sensing. Gawad, et al. Lab Chip 1; 76 (2001);Lee, et al. Proceedings of the 18th IEEE International Conference onMicro Electro Mechanical Systems (MEMS) 678-681 (2005); Satake et al.Sens. Actuators B: Chem. 83; 77 (2002); Morgan, et al. Curr. Appl. Phys.6, 367-370 (2006); Lee et al., J. Micromech. Microeng. 15; 447-454(2005); Altendorf, et al. Proceedings of the International Conference onSolid State Sensors and Actuators (Transducers '97) v. 1, p. 531,Chicago, Ill. (1997); Holmes et al., Biosens. Bioelectron. 21; 1621-1630(2006); Yang et al., Meas. Sci. Technol. 17; 2001-2009 (2006); Simonnetet al., Anal. Chem. 78; 5653-5663 (2006); Niehren, et al., Anal. Chem.67; 2666-2671 (1995).

In the area of optical sensing, microfabrication has allowed developmentof microdevices to replace glass capillary-based flow chambers, and tointegrate compact optics and provide on-chip sample transport.

Cell sensing and counting, particularly of leukocytes, is cumbersome duein part to the cell population numbers. For leukocyte differential inmicrodevices based on optical sensing, a V-groove micro-channel wasfabricated by anisotropic wet etching of a silicon substrate and 3-partleukocyte differential was demonstrated for diluted blood without sheathflow by two-parameter light scattering. Altendorf, Proceedings of theInt'l Conference on Solid State Sensors and Actuators, v. 1, p. 531(1997).

However, until the instant embodied disclosure, it was necessary todilute cell samples for cell sensors and actuators for many reasons. Onereason dilution has been necessary is in order to prevent thecoincidence effect in which multiple cells appear in the defection zonesimultaneously. In human blood, the ratio of erythrocytes, or red bloodcells, to leukocytes is on the order of about a thousand to one, adilution factor of from about one hundred to several tens of thousandsis typically required to avoid erythrocyte interference for electricalimpedance or light scattering defection. Furthermore, for countingleukocytes in samples where leukocytes are specifically fluorescentlylabeled, a dilution of at least ten times is usually required. Sheenanand Storey, J. Pathol. Bacteriol. 59; 336 (1947); Kass, J. Clin. Pathol.76; 810-12 (1981); Weigl et al., Biomed. Microdev. 3; 267-274 (2001);

Dilution is also often required in order to avoid clogging samplechambers, and also In order to remove erythrocytes that are lysed priorto running the sample, particularly for electrical impedance or lightscattering detection. Some of these protocols also require an additionalfixation buffer.

Dyes

In the present disclosure, a dye, such as Acridine orange(3,6-dimethylamineoacridine, FIG. 1), can be used to differentiate atarget, such as cells, organelles, granules, nuclei, molecules(including double or single stranded nucleic acids, such as DNA, or RNA,chromosomes, and also including synthetic forms). In one particularembodiment, leukocytes may be detected, counted, or sorted without needfor lysing erythrocytes or fixing the cell sample. Certain dyes, such asAcridine orange, are also desirable due to the fast diffusion intocells, easy commercial availability, and excitation and emissionwavelength compatibility with common light sources (i.e. argon laser andother broad spectrum light sources in visible range) and opticalfilters. Kosenow, Acta Haematol. 7, 217 (1952); Schiffer, Blood, 19, 200(1962): Jackson, Blood, 17, 643 (1961); Hallermann et al., Verh DeutschGes Inn Med. 70, 217 (1964).

Acridine orange is a pH-sensitive fluorescent cationic dye that binds todouble-stranded DNA by electrostatic attraction and intercalation of theAcridine orange between base pairs. Upon binding, the excitation maximumbecomes 502 nm and the emission maximum becomes 525 nm (green). Acridineorange also binds to RNA and single-stranded DNA, with a shiftedexcitation maximum of 460 nm and an emission maximum of 650 nm (red).Adams and Kamentsky, Acta Cytol. 15, 289 (1971); Adams and KamentskyActa Cytol. 18, 389-91 (1974); Steinkam et al., Acta Cytol. 17, 113-17(1973). Acridine orange is also desirable in that it is hydrophobic inneutral pH, and can easily diffuse through the cell membrane and cellnuclear membrane to bind to RNA and DNA. In living cells, Acridineorange is protonated in the acidic environment of lysosomes, which makesit cationic and prevents the dye from leaking out of lysosome membranes.Moriyama et al., J. Biochem. 92; 1333-36 (1982). When Acridine orange isused for leukocyte analysis, the cell nucleus is stained green withslightly mixed red, a result of double-stranded DNA and single-strandedRNA, while the cell cytoplasm is stained red due to the RNA andlysosomes. Thus, leukocyte counting can be achieved easily by using thestrong signal from the green fluorescent channel. Leukocytedifferentiation can be achieved by analyzing the signal from the redfluorescence channel.

For fresh-stained leukocytes, a 3-part differential (lymphocytes,monocytes, and granulocytes) can be achieved by studying the redfluorescent signal of an Acridine orange stained diluted blood sample,whereas a 5-part differential leukocytes (lymphocytes, monocytes,neutrophils, eosinophils, and basophils) has been demonstrated withhypotonic dilution and fresh Acridine orange-stained leukocyte samples.Adams and Kamentsky, Acta Cytol. 15, 289 (1971); Adams and Kamentsky,Acta Cytol. 18, 389-391 (1974); Steinkam et al., Acta Cytol. 17, 113-17(1973).

Other dyes can be utilized with certain embodiments described in theinstant disclosure, such as ethidium bromide, three-dye combinations(ethidium bromide, brilliant sulfaflavine, and stilbene disulfonic acidderivative); oxazine dyes, basic orange 21, and a polymethine dye.Shapiro, et al., J. Histochem. Cytochem. 24, 396-411 (1976); Shapiro, etal., J. Histochem. Cytochem. 25, 836-844 (1977); U.S. Pat. No.4,376,820; U.S. Pat. No. 4,882,284; Tibbe, et al., Nat. Biotechnol. 17,1210-1213 (1999); U.S. Pat. No. 4,400,370; Kass, J. Histochem. Cytochem.36, 711-715 (1988).

Apparatus

One embodiment of the instant disclosure relates to a device orapparatus for cell counting and/or differentiating. In particularembodiments, the device or apparatus comprises a substrate formed from amaterial, such as silicon, glass, plastic, metal, or other material. Oneparticular embodiment of the instant disclosure was fabricated usingsoft lithography. Quake, Science 290, 1536-40 (2000). Otherphotolithographic or etching techniques could also be used, according tospecific embodiments.

One embodiment of the device was microfabricated using two parts of PDMS(polydimethylsiloxane) (Sylgard 184, Dow Corning, MI, USA) mixedvigorously in 10:1 ratio. After degassing in vacuum for about 30minutes, the mixture was poured onto DRIE-etched silicon mold, that hadbeen pretreated with HMDS (hexamethyldisilazane) for easy separationafter baking. The molds were baked at 80° C. for 30 minutes. Thehardened PDMS was separated from the silicon mold, and PDMS sheet wascut into pieces and fluidic access holes were punctured on each piecewith a Luer stub adapter (Becton Dickinson, NJ, USA). Each PDMS piecewas carefully placed on a cleaned glass slide and baked overnight at 80°C. In some cases, oxygen plasma treatment (300 m Torr, 25 W, 30 s) wasused for PDMS and glass slides in order to improve adhesion betweenthem, particularly with devices that were intended to be reused.Bhattacharya et al., J. Microelectromechan. Syst. 14, 590-97 (2005).

In one particular embodiment, the channel structure was molded on a 1cm×1 cm PDMS block, with the thickness of the PDMS block at less than 3mm. In one particular embodiment the channel depth was 16 μm in order toaccommodate large leukocyte sizes.

One exemplary embodiment of the device is shown in FIG. 2. For thisparticular embodiment, a first fluid flow Inlet 200 allows fordeposition of, for example sheath flow fluid, and is in fluidcommunication with a bifurcated channel with a first channel arm 260 anda second channel arm 270 that both converge at a junction of a reservoir290 and the detection zone 240. In this particular embodiment, theapparatus further comprises a second fluid flow inlet 210 that allowsfor deposition of, for example, a sample fluid, such as blood, that isin fluid communication with a filter array structure 230, by way of abranched sample flow zone channel 220 and a fluid flow outlet 250. Inthis particular exemplary embodiment, 2-D hydrodynamic focusing wasadopted to control the particle position of the cell sample in thedetection zone 240. According to the embodiment shown in FIG. 2, theratio of cross-sectional area of sheath flow to core sample flow was10:1, and the channel width of the defection zone 240 was 50 μm, withthe width of the focused sample flow preferably 5 μm or less. Inparticular embodiments, the channels comprise a physical feature, suchas a depression or a protrusion.

One other exemplary embodiment of the device is shown in FIG. 13A. Forthis particular embodiment the fluid flow inlet 1340 allows fordeposition of a sample fluid, such as a biological sample, or otherfluid sample containing a target. In one particular embodiment, thebiological sample includes a cell sample, such as blood. In thisexemplary embodiment, the fluid inlet is in fluid communication with afirst channel 1330 which contains a restrictive access 1320 that isjuxtaposed to a second channel 1310 which comprises the detection zonewhich is also in fluid communication with the fluid flow outlet 1300. Incertain embodiments, the height of the first and/or second channels isapproximately 5 μm, approximately 8 μm, approximately 10 μm,approximately 12 μm, approximately 15 μm, approximately 20 μm,approximately 25 μm, approximately 30 μm, approximately 35 μm,approximately 40 μm, or any value therebetween. In certain embodiments,the width of the second channel is approximately 5 μm, 10 μm,approximately 15 μm, approximately 20 μm, approximately 25 μm,approximately 30 μm, approximately 35 μm, approximately 40 μm,approximately 45 μm, approximately 50 μm, or any value therebetween. Inthe exemplary embodiment shown in FIG. 13A, the second channel width wasapproximately 20 μm in size.

One other exemplary embodiment of the device is shown in FIG. 13B. Forthis particular embodiment, the fluid flow inlet 1440 allows fordeposition of a sample fluid, such as a biological sample, or otherfluid sample containing a target. In one particular embodiment, thebiological sample includes a cell sample, such as blood. In thisexemplary embodiment, the fluid inlet is in fluid communication with afirst channel 1430 which contains a restrictive access 1420 that isjuxtaposed to a second channel 1410 which comprises the detection zonewhich is also in fluid communication with the fluid flow outlet 1400. Incertain embodiments, the height of the first and/or second channels isapproximately 5 μm, approximately 8 μm, approximately 10 μm,approximately 12 μm, approximately 15 μm, approximately 20 μm,approximately 25 μm, approximately 30 μm, approximately 35 μm,approximately 40 μm, or any value therebetween. In certain embodiments,the width of the second channel is approximately 5 μm, 10 μm,approximately 15 μm, approximately 20 μm, approximately 25 μm,approximately 30 μm, approximately 35 μm, approximately 40 μm,approximately 45 μm, approximately 50 μm, or any value therebetween. Inthe exemplary embodiment shown in FIG. 13B, the second channel width wasapproximately 30 μm in size.

Certain embodiments of the device use a focused laser source forillumination, since cell focusing in the detection zone 240 is highlydesirable. However, other embodiments included in the present disclosureuse a more uniform diffused light source and a slit aperture. Suchembodiments utilize straight channel geometry without cell focusing. Inone embodiment, the channel length of the detection zone 240 is 1000 μm.A filter structure 230 upstream of the sample flow zone 220 may also beincluded in certain embodiments, which filtered out contaminants,including erythrocyte rouleaux, and other large particle aggregates toprevent clogging in the detection zone 240. In certain embodiments, thesize of the rectangular pillar structure components of the filterstructure 230 was 200 μm×40 μm. The spacing between the pillars in eachof the three rows was 40 μm, 30 μm, and 20 μm respectively, which allowsfor even the largest leukocytes to pass through the filter region 230.

System

The optical system was set up on an optical bench as shown in FIG. 3(transmitted laser-induced fluorescent detection system or LIF). In oneparticular embodiment, the system setup comprises an excitation or lasersource 300, a lens assembly 340, the microfluidic apparatus 350, anoptional additional lens assembly 360, a filter assembly 320, 330, andan image capture device 390, 395. In certain embodiments, one or moreemission filters comprise, the filter assemblies 320, 330. In certainembodiments, the image capture device 395 comprises a charge coupleddevice (CCD) camera, a complementary metal-oxide-semiconductor (CMOS)device, or a photomultiplier tube (PMT) device. In particularembodiments, the image capture device 395 may be coupled to communicatewith a display unit or computing device 396, such as a personalcomputer. One of skill in the art would recognize that multiple andvarious computer software programs are available that allow forintegration, compilation, analysis, reconfiguration, and othermanipulation of data received from the system, particularly by way ofthe computing device 396.

In one particular exemplary embodiment, an argon laser (National LaserNLC210BL, 483 nm, and 15-30 mW adjustable, Salt take City, Utah, USA) isused as the excitation source. An aperture 310 of 50 μm diameter is putin front of the laser output to facilitate the alignment process andlower the illumination intensity. In certain embodiments, art optionallaser-line bandpass filter (bandwidth equal to about 1.9 nm with acentral wavelength of 488 nm) is used to further purify the lasersource. In certain other embodiments, an optional neutral density filter(NDF) is used to attenuate laser excitation. Alternatively, the pinholeand NDF are replaced by two linear glass polarizers (Edmond Optics TECHSPEC, Barrington N.J., USA) so that the Illumination level on the devicecan be easily adjusted.

In one particular embodiment, a long-working-distance microscopeobjective (USMCO M Plan Apo, 10×, 0.28 NA, Dayton, Nev., USA) is used asa condenser lens 340. Another long-working-distance microscope objective(Bausch & Lomb, 50×, 0.45 NA, Rochester, N.Y., USA) is used as anobjective lens 360. In the same embodiment, three emission filters 320are used in one particular test: 488 nm long pass filter (Chroma H1500LP, transition width>4.9 nm, edge steepness=2.5 nm, Rockingham, Vt.,USA), a green bandpass filter with central wavelength 525 nm and abandwidth 50 nm (Chroma D525_50 m), and a red bandpass filter withcentral wavelength 650 nm and bandwidth 50 nm (Chroma D650_50 m). Abroadband non-polarizing hybrid cube beamsplitter 370 (Newport05BC17MB.1, 400-700 nm, R/T=45%/45%, Irvine, Calif., USA) is used todirect light to the photodiode detector 380 and CCD camerasimultaneously.

The signal is electrically amplified and detected either with a siliconphotodiode receiver module 390 (Electro-Optical Systems, UVS-025-H,Phoenixville, Pa., USA) or a photon multiplier tube (PMT, HamamatsuH5784-20, Japan). The voltage signal is sent to a deep memoryoscilloscope (HP 54645A, Palo Alto, Calif., USA). When the buffer in theoscilloscope is full, the data can be loaded to a computer and analyzedwith a Matlab peak-detection program. Video may be taken with an analogCCD camera (Hitachi KP-D20B, Japan) at 30 frames per second and thenconverted to digital format and stored in a computer 396. Other imagingcapture devices 395, such as CMOS, PMT, or still other devices may alsobe used with particular embodiments described herein. In certainexemplary test runs, the system set up utilizing a photodiode detectorand PMT are more sensitive than the CCD camera and have a faster timeresponse. During one exemplary test run, the optical system was firstroughly aligned on a dummy device with the aid of images from CCDcamera. A 10 μm diameter illumination spot on the detection zone iseasily achieved with proper alignment.

As shown in FIG. 12A and FIG. 12B, the instant apparatus may beincorporated into a hand-held unit comprising a laser source (such as alaser emitting diode or LED 120), at least one lens 190, at least onefilter assembly with optional beamsplitter 195, a microfluidic apparatusas described herein on a microchip or other substrate 185, aninput/output port 130, at least one image capture device 100, 110, whichmay be a photomultiplier tube. In certain embodiments, the hand-heldunit may be assembled and enclosed by an outer casing or casings 160,180, and rivets or bolts 140, 160.

Cell Detection

One aspect of the instant disclosure relates to methods of countingand/or differentiating cells, particularly leukocytes, from undilutedcell samples, such as human or other animal blood, by utilizingmicrofabricated devices. In one exemplary embodiment, cell detection wasconducted utilizing Acridine orange and fresh whole human blood.

In one exemplary embodiment, fresh human blood was obtained from healthydonors and used within 3 days of collection. EDTA was added to the bloodsamples in order to prevent coagulation. For Acridine orange staining,the stock solution was added to obtain a final dye-concentration of 10μg/mL in Ficoll-Paque Plus. Ficoll-Paque Plus was also used as thesheath flow solution. Fluorescent polystyrene beads (5 μm greenfluorescent beads) were purchased from Duke Scientific Corporations,Fremont, Calif., USA. Cell nucleus stain Acridine orange was obtainedfrom Molecular Probes, Eugene, Oreg., USA, and dissolved in water toachieve a 10 mg/mL stock solution. Blood diluent Ficoll-Paque Plus waspurchased from Amersham Biosciences, Sweden. Phosphate buffered saline(10×PBS) was obtained from Ambion (9625), Austin, Tex., USA.

Staining results were observed under a fluorescent microscope (NikonE800, Japan) with a triple band filter block DAPI-FITC-TRITC, which hasexcitation filter wavelengths of 385-400 nm, 475-490 nm, and 545-565 nm,and emission filter wavelengths of 450-465 nm, 505-535 nm and 580-620nm. Images were taken with a cooled CCD camera (RT-KE color 3-shot,Diagnostic Instruments, Sterling Heights, Mich., USA). Rough count ofleukocytes was made with a hemactyometer (Hausser Scientific, Horsham,Pa., USA). When necessary, blood or fluorescent beads were diluted withFicoll-Paque Plus (specific gravity 1.077 g/mL) to match the specificgravity of the solvent to leukocytes. All fluids were pumped into thedevices using syringe pumps (Harvard Apparatus Pico Plus, Holliston,Mass., USA).

In this particular embodiment, an analog CCD camera was used for videorecording at a matched camera frame rate of 3 nL/min sample flow rateand 30 nL/min sheath flow rate. For photodiode detection, a 0.1μL/minute sample flow rate and a 1 μL/minute sheath flow rate were used.A 1 μL/minute sample flow and a 10 μL/minute sheath flow were used withthe photon multiplier tube instrument.

In order to achieve a high signal-to-noise ratio, the maximalconcentration for cell staining was established using routine methods inthe art. Adams and Kamentsky, Acta Cytol. 15; 289 (1971). As shown inFIG. 4, whole blood samples were analyzed with different Acridine orangeconcentrations. The optimal concentration for leukocyte staining wasdetermined to be approximately in the range of 1 μg/mL. In theparticular exemplary embodiment utilized in FIG. 4, the distance betweenthe coverslip and the grid surface was approximately 100 μm. As can beseen in FIG. 4A, an abundance of erythrocytes were present under thefield of view, yet these cells did not interfere with the fluorescentsignal from the leukocytes, as shown in FIG. 4B-F.

As can be seen in FIG. 5, the exemplary embodiment utilized in celldetection did not experience any significant photobleaching. The signalwas fitted as a first-order exponential decay with time constant of6.4+/−0.7 seconds. Two more tests confirmed that the photobleaching timeconstant for one particular embodiment was between 1 second and 10seconds. The photobleaching time constant for one particular embodimentwas characterized by filling the device with Acridine orange-stainedwhole blood. The channel was scanned by the laser spot and theillumination was set to be the same as that used in testing. The entireprocess was recorded with a CCD camera. Whenever a fluorescing leukocytewas observed with fluorescent emission clearly distinct from thebackground, we stopped moving the laser spot and waited until theleukocyte was photobleached to background level. The images wereextracted from the video, converted to 8-bit gray scale images, andanalyzed with a Matlab program. The data was fitted to a singletime-constant exponential decay.

Additionally, green fluorescent beads were tested at a concentration ofabout 2×10³/μL, as observed by CCD camera, and shown in FIG. 6. Sampleflow rate was set at about 3 nL/min, and sheath flow was about 30nL/min. In one exemplary test run, a hydrodynamic focused laser beam, asshown in FIG. 6A, created an enlarged light circle as shown in FIG. 6B.Only a single bead normally appeared in each image. With diffused laserillumination, as shown in FIG. 6C, the trace of the bead could beidentified, as shown in FIG. 6D. Hydrodynamic focusing limits thecross-sectional area of the defection zone without shrinking the channeldiameter, thus the signal-to-noise ratio may be improved withoutincreasing the risk of clogging the channel. Also, the reduction of thecross-section of the core flow reduces the coincidence effect. Finally,enclosing the core sample flow with sheath flow minimizes fluorescentdye absorption in the device walls, thus reducing background noise. Asindicated in FIG. 7, bead signals from the photodiode detector couldeasily be identified.

As shown in FIG. 8, using both red and green emission filters, imagesextracted from video taken by the CCD camera show the signal identifiedfrom a leukocyte stained with Acridine orange, as well as the signalobtained from the fluorescent control bead. For photodiode detection,the expected leukocyte detection rate would average about 4-11 cells persecond for a normal individual.

In one exemplary embodiment, a time trace over 50 seconds of anundiluted blood sample stained with Acridine orange using a greenemission filter, and a throughput of up to about 1000 leukocytes persecond was attained. Maxima signal Intensity (peak height as in FIG. 9)from the green fluorescent channel with 525 nm emission filter wasstudied by plotting its histogram, as shown in FIG. 10. As expected, thelower-intensity portion Is likely contributed mainly by lymphocytes,while the higher-intensity portion is likely mainly from monocytes, withthe center-region is likely mostly from granulocytes. Steinkam et al.,Acta Cytol. 17; 113-117 (1973).

In one exemplary embodiment, a time trace over 50 seconds of anundiluted blood sample stained with Acridine orange using a redfluorescent channel with 650 nm emission filter was conducted. As shownin FIG. 11, two peaks were identified. The lower intensity is dominatedby lymphocytes and the higher-intensity peak is largely monocytes andgranulocytes. The time between the start of staining the cells tophotodiode recording was typically greater than 15 minutes.

In both exemplary studies, the maximal throughput was about 1000leukocytes per second utilizing one embodiment of the PMT detector. Byusing undiluted blood, minimal sample volume was maintained, whichincreases the throughput. Since sample throughput is proportional tovolume flow rate, but is limited by the maximal pumping rate andresponse time of the sensing system, a 3 nL/minute core flow rate wasused with the CCD camera defection. Under this flow rate, a typicalleukocyte traveled through the detection zone in approximately 30milliseconds, which roughly equals the CCD frame acquisition time.

Flow rates for varying embodiments may be suitable for a range fromapproximately 1 nL/minute, approximately 2 nL/minute, approximately 3nL/minute, approximately 4 nL/minute, approximately 5 nL/minute,approximately 6 nL/minute, approximately 7 nL/minute, approximately 8nL/minute, approximately 9 nL/minute, approximately 10 nL/minute,approximately 20 nL/minute, approximately 30 nL/minute, approximately 40nL/minute, approximately 50 nL/minute, approximately 60 nL/minute,approximately 70 nL/minute, approximately 80 nL/minute, approximately 90nL/minute, approximately 100 nL/minute, approximately 110 nL/minute,approximately 120 nL/minute, approximately 130 nL/minute, approximately140 nL/minute, approximately 150 nL/minute, or any value therebetweenfor photodiode detection. Likewise, for PMT defection, flow rates forvarying embodiments may be suitable for a range from approximately 200nL/minute, approximately 300 nL/minute, approximately 400 nL/minute,approximately 500 nL/minute, approximately 600 nL/minute, approximately700 nL/minute, approximately 800 nL/minute, approximately 900 nL/minute,approximately 1 μL/minute, approximately 2 μL/minute, approximately 3μL/minute, approximately 4 μL/minute, approximately 5 μL/minute, or anyvalue therebetween.

In one exemplary embodiment, the time response of the photodiodereceiver module under low sensitivity setting was 0.16 milliseconds, and0.6 milliseconds under high sensitivity, while the time response of thePMT detector in one exemplary run was about 16 microseconds.

Furthermore, by decreasing the cross-sectional area, the linear flowvelocity of the core flow is increased, which requires faster sensing,and reduces the coincidence effect by increasing the average distancebetween cells in the detection zone.

Thus, by utilizing particular embodiments disclosed herein relating to amicrofluidic device, leukocyte sensing, counting, and sorting can beachieved one-by-one in a micro flow cytometer system. Furthermore, densecell suspensions, such as whole, undiluted blood may be utilized incertain embodiments described herein, which provides for reduced sampleand waste volume, reduced processing time, and completely eliminateson-chip mixing and buffer storage. In particular aspects, leukocytes canbe sensed one-by-one in a micro flow cytometer system.

As described herein, certain embodiments of the device can beimplemented in various sizes and conformations, including but notlimited to a bench-top device, a handheld device (such as is shown inFIG. 12), an implantable device, a nanotechnology device, or other sizeor conformation. In the smaller exemplary conformations,high-illumination LED is used for excitation and a minipump is used tomanipulate the sample in suction mode, while fluorescent signals fromgreen and red channels can be detected simultaneously.

INCORPORATION BY REFERENCE

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

The invention claimed is:
 1. A method for analyzing cells, the methodcomprising: (a) introducing a fluid sample comprising fluorescentlylabeled cells and fluorescently labeled control beads to an inlet of afirst microfluidic channel, wherein the fluorescently labeled cells arelabeled with a fluorophore different than a fluorophore label of thecontrol beads; (b) flowing the fluid sample from the inlet of the firstmicrofluidic channel to an outlet of a second microfluidic channel,wherein an outlet of the first microfluidic channel is physically andfluidically coupled to an inlet of the second microfluidic channel andwherein the flow of the fluid sample is aided with a pump; (c) excitingthe fluorophores of the fluorescently labeled cells and of thefluorescently labeled control beads using an excitation source; (d)detecting fluorescence of the fluorescently labeled cells andfluorescence of the fluorescently labeled control beads as they passthrough a detection zone within the second microfluidic channel, whereinthe detection is performed on individual cells; (e) analyzing thedetected fluorescence of the fluorescently labeled cells andfluorescence of the fluorescently labeled control beads using a computersoftware inscribed on a computing device; and (f) providing a resultconcerning one or more properties of the cells being analyzed.
 2. Themethod of claim 1, wherein the cells are leukocytes.
 3. The method ofclaim 1, wherein the fluid sample is a treated blood sample.
 4. Themethod of claim 1, wherein the second microfluidic channel has fixeddimensions.
 5. The method of claim 4, wherein the fixed dimensions areof approximately 40 microns by approximately 50 microns.
 6. The methodof claim 1, wherein the first channel and the second channel areobstacle-free.
 7. The method of claim 1, wherein the flowing of thefluid sample from the inlet of the first microfluidic channel to theoutlet of the second microfluidic channel increases a core flow rate ofthe fluid sample to form an increased flow rate.
 8. The method of claim1, wherein the method further comprises identifying the fluorescentlylabeled cells.
 9. The method of claim 1, wherein the method furthercomprises sorting the fluorescently labeled cells.
 10. The method ofclaim 1, wherein the method further comprises counting the fluorescentlylabeled cells.
 11. The method of claim 1, further comprising videorecording a measurement on a recording unit.
 12. The method of claim 1,wherein the method can detect cells at a rate of up to about 1000 cellsper second.
 13. The method of claim 1, wherein the excitation source isa laser or a LED.
 14. The method of claim 1, wherein the excitationsource comprises a lens assembly.
 15. The method of claim 1, furthercomprising manipulating the detected fluorescence using a computersoftware program.
 16. The method of claim 15, further comprisingdisplaying a result of the manipulation on a display unit.
 17. Themethod of claim 1, wherein the fluorescently labeled beads are polymericbeads.
 18. The method of claim 1, wherein the fluorescently labeledbeads are polystyrene beads.
 19. The method of claim 1, wherein the stepof detecting fluorescence comprises using an image capture device. 20.The method of claim 1, wherein flowing the fluid sample comprisespumping the fluid sample through a microfluidic device with the pump.21. The method of claim 1, wherein the fluid sample does not comprise asheath fluid.
 22. The method of claim 1, wherein the fluid sample is anundiluted blood sample.